DESCRIPTION: (Applicant's Abstract) Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) constitute the primary mechanism for inactivation of 5HT and are high-affinity targets for important addictive and therapeutic drugs including MDMA, fenfluramine, cocaine, tricyclics and SSRIs. In our previous funding period, we and collaborators cloned human, mouse and Drosophila melanogaster SERT cDNAs, mapped the human gene to chromosome 17q11.2, mapped protein contact sites for SERT antagonists including tricyclics, mazindol and citalopram, identified kinase-linked pathways supporting acute hSERT regulation, replicated SERT regulation under voltage-clamp and demonstrated polarized surface expression of SERT proteins. Our goal in this renewal application is to more completely understand how SERTs are organized at the plasma membrane and how transport is modulated by extracellular signals and intracellular signaling pathways. Our published and preliminary studies reveal kinase and phosphatase-dependent modulation of SERT transport capacity, SERT-regulatory trafficking pathways, 5HT-modulated phosphorylation states, and SERT-associated proteins. In this renewal, we evaluate the structural requirements for SERT membrane expression, phosphorylation and sequestration, evaluate whether SERT phosphorylation is required for acute transporter regulation, and establish the structural and functional basis of interactions with SERT-associated proteins. Using patch clamp techniques, we will explore the possibility that SERTs exist in the plasma membrane associated with regulatory enzymes that can modify transport activity prior to, or as a requirement for, sequestration. These studies will also shed light on how SERT functional expression is controlled by presynaptic hetero- and autoreceptors and transport-modulatory ligands, including the amphetamines and 5HT itself. Our studies offer opportunities for new insights into mechanisms of drug action and may uncover strategies for the design of novel therapeutics that manipulate amine transporter reulation rather than interfering with 5HT recognition. SERT homologies with other gene family members, including NETs and DATs, suggests that advances made with SERTs may have heuristic and practical benefits for the study of other neurotransmitter transporters linked to mental illness and drug abuse.